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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester
Physical state/Appearance: White solid
CAS Number: 78433-08-6
Purity: >98%
Storage Conditions: Room temperature in the dark
Analytical monitoring:
Details on sampling:
Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in tetrahydrofuran to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with tetrahydrofuran to produce a stock solution of 500 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 0.50 mg/L. Five replicates at this concentration level were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.
Details on test solutions:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3
Test temperature:
21C to 22C
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary)
Dissolved oxygen:
Aerated until the dissolved oxygen concentration was approximately air-saturation value.
Nominal and measured concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give the 100% v/v saturated solution test concentration.
Details on test conditions:
A150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period.
Reference substance (positive control):
potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Key result
48 h
Dose descriptor:
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
There was no immobilization in 20 daphnids exposed to a test concentration of 100% v/v saturated solution for a period of 48 hours.

Exposure of Daphnia magna to the test item gave EC50 values based on nominal test concentrations of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.

No sub-lethal effects of exposure were observed throughout the test.
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

 Range-finding Test

No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at highest attainable test concentration, no immobilization or adverse reactions to exposure were observed.

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.019 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Validity criteria fulfilled:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the nominal test concentrations gave a 48-Hour EC50 value of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.

Description of key information

In an OECD 202 study, under GLP conditions, the 48 hour EC50 to freshwater invertebrates (Daphnia magna) of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester has been investigated is greater than 100% v/v saturated solution. The No Observed Effect Concentration is 100% v/v saturated solution (Envigo, 2016f).


Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.019 mg/L

Additional information